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745100.9969Plant species influences the composition of root system microbiome and its antibiotic resistance profile in a constructed wetland receiving primary treated wastewater. INTRODUCTION: Constructed wetlands (CWs) are nature-based solutions for wastewater treatment where the root system microbiome plays a key role in terms of nutrient and pollutant removal. Nonetheless, little is known on plant-microbe interactions and bacterial population selection in CWs, which are mostly characterized in terms of engineering aspects. METHODS: Here, cultivation-independent and cultivation-based analyses were applied to study the bacterial communities associated to the root systems of Phragmites australis and Typha domingensis co-occurring in the same cell of a CW receiving primary treated wastewaters. RESULTS AND DISCUSSION: Two endophytic bacteria collections (n = 156) were established aiming to find novel strains for microbial-assisted phytodepuration, however basing on their taxonomy the possible use of these strains was limited by their low degrading potential and/or for risks related to the One-Health concept. A sharp differentiation arose between the P. australis and T. domingensis collections, mainly represented by lactic acid bacteria (98%) and Enterobacteriaceae (69%), respectively. Hence, 16S rRNA amplicon sequencing was used to disentangle the microbiome composition in the root system fractions collected at increasing distance from the root surface. Both the fraction type and the plant species were recognized as drivers of the bacterial community structure. Moreover, differential abundance analysis revealed that, in all fractions, several bacteria families were significantly and differentially enriched in P. australis or in T. domingensis. CWs have been also reported as interesting options for the removal of emerging contaminants (e.g, antibiotic resistance genes, ARGs). In this study, ARGs were mostly present in the rhizosphere of both plant species, compared to the other analyzed fractions. Notably, qPCR data showed that ARGs (i.e., ermB, bla (TEM), tetA) and intl1 gene (integrase gene of the class 1 integrons) were significantly higher in Phragmites than Typha rhizospheres, suggesting that macrophyte species growing in CWs can display a different ability to remove ARGs from wastewater. Overall, the results suggest the importance to consider the plant-microbiome interactions, besides engineering aspects, to select the most suitable species when designing phytodepuration systems.202439113842
354810.9969From flagellar assembly to DNA replication: CJSe's role in mitigating microbial antibiotic resistance genes. The emergence of Antibiotic Resistance Genes (ARGs) in Campylobacter jejuni (CJ) poses a severe threat to food safety and human health. However, the specific impact of CJ and its variants on ARGs and other related factors remains to be further elucidated. Herein, integrated metagenomic sequencing and co-occurrence network analysis approach were employed to investigate the impact of CJ and CJ incorporated with biogenic selenium (CJSe) on ARGs, flagellar assembly pathways, microbial communities, and DNA replication pathways in chicken manure. Compared to the Control (CON) and CJ groups, the CJSe group exhibited 2.4-fold increase selenium levels (P < 0.01) in chicken manure. Notable differences were also observed between the CJ and CJSe groups, with sequence results showing a CJ > CJSe > CON trend in total ARG copy numbers. Furthermore, the CJSe group showed 31.6 % fewer flagellar assembly genes compared to the CJ group. Additionally, compared to the CJ group, CJSe inhibited pathways such as basal body/hook (e.g., FliH, FliO, FliQ reduced by 25-52 %) and stator (MotB downregulated by 42.3 %), suppressing flagellar assembly. We also found that both CJ and CJSe influenced bacterial DNA replication pathways, with the former increasing ARG-carrying bacteria and the latter, under selenium-induced selective pressure, reducing ARG-carrying bacteria. Moreover, compared to the CJ group, the CJSe group showed a significantly lower 9.72 % copy number of total archaeal DNA replication genes. Furthermore, through intricate co-occurrence network analysis, we discovered the complex interplay between changes in ARGs and bacterial and archaeal DNA replication dynamics within the microbial community. These findings indicate that CJSe mitigates the threat posed by CJ and reduces ARG prevalence, while its dual functionality enables applications in biofortified crop production and soil remediation in selenium-deficient regions, thereby advancing circular economy systems. While the current study demonstrates CJSe's dual functionality under controlled conditions, future work will implement a dedicated ecological risk assessment framework encompassing Se speciation/leaching tests and non-target organism assays to confirm environmental safety under field-relevant scenarios. This approach aligns with sustainable strategies for food security and public health safeguarding.202541108960
683320.9967Comprehensive insights into profiles and bacterial sources of intracellular and extracellular antibiotic resistance genes in groundwater. Antibiotic resistance genes (ARGs), especially last-resort ARGs (LARGs), are receiving extensive attention as emerging environmental contaminants in groundwater. However, their prevalent intracellular and extracellular patterns and bacterial sources in groundwater remain unclear. Herein, groundwater samples were collected in Tianjin, and characterized based on the profiles of intracellular ARGs (iARGs) and extracellular ARGs (eARGs), as well as the resident bacterial communities and extracellular DNA (eDNA)-releasing bacterial communities. The quantitative real-time PCR assays showed that eARGs presented fewer subtypes than iARGs and generally displayed lower detection frequencies than the corresponding iARGs. Similarly, LARGs exhibited lower detection frequencies than common ARGs, but the total abundance showed no significant differences between them. Genes vanA and bla(VIM) were the observed dominant LARGs, and aadA was the observed common ARG independent of location inside or outside the bacteria. Furthermore, the top 10 phyla showed much difference between the main eDNA-releasing bacteria and the dominant resident bacteria. Proteobacteria was the predominant resident bacterial phyla while dominating the source of eDNA in groundwater. Despite representing a minor portion of the abundance in the resident bacteria, Actinobacteriota, Acidobacteriota, and Chloroflex surprisingly accounted for a large majority of eDNA release. Co-occurrence patterns among persistent ARGs, the resident bacteria, and eDNA-releasing bacteria revealed that the dominant common iARG aadA and intracellular LARGs bla(VIM) and vanA had significant positive correlations with Methylobacterium_Methylorubrum and Shewanella. Meanwhile, the dominant extracellular LARG bla(VIM) may be released by bacteria belonging to at least five genera, including Ellin6067, Bifidobacterium, Blautia, Veillonella, and Dechloromonas. Collectively, the findings of this study extend our understanding regarding the distribution of ARGs and their bacterial sources in groundwater, and indicate the serious pollution of LARGs in groundwater, which poses potential risks to public health.202235623567
865430.9967Metagenomic and Metatranscriptomic Study of Microbial Metal Resistance in an Acidic Pit Lake. Cueva de la Mora (CM) is an acidic, meromictic pit lake in the Iberian Pyrite Belt characterized by extremely high metal(loid) concentrations and strong gradients in oxygen, metal, and nutrient concentrations. We hypothesized that geochemical variations with depth would result in differences in community composition and in metal resistance strategies among active microbial populations. We also hypothesized that metal resistance gene (MRG) expression would correlate with toxicity levels for dissolved metal species in the lake. Water samples were collected in the upper oxic layer, chemocline, and deep anoxic layer of the lake for shotgun metagenomic and metatranscriptomic sequencing. Metagenomic analyses revealed dramatic differences in the composition of the microbial communities with depth, consistent with changing geochemistry. Based on relative abundance of taxa identified in each metagenome, Eukaryotes (predominantly Coccomyxa) dominated the upper layer, while Archaea (predominantly Thermoplasmatales) dominated the deep layer, and a combination of Bacteria and Eukaryotes were abundant at the chemocline. We compared metal resistance across communities using a curated list of protein-coding MRGs with KEGG Orthology identifiers (KOs) and found that there were broad differences in the metal resistance strategies (e.g., intracellular metal accumulation) expressed by Eukaryotes, Bacteria, and Archaea. Although normalized abundances of MRG and MRG expression were generally higher in the deep layer, expression of metal-specific genes was not strongly related to variations in specific metal concentrations, especially for Cu and As. We also compared MRG potential and expression in metagenome assembled genomes (MAGs) from the deep layer, where metal concentrations are highest. Consistent with previous work showing differences in metal resistance mechanisms even at the strain level, MRG expression patterns varied strongly among MAG populations from the same depth. Some MAG populations expressed very few MRG known to date, suggesting that novel metal resistance strategies remain to be discovered in uncultivated acidophiles.202032899650
335140.9967Quantification of the mobility potential of antibiotic resistance genes through multiplexed ddPCR linkage analysis. There is a clear need for global monitoring initiatives to evaluate the risks of antibiotic resistance genes (ARGs) towards human health. Therefore, not only ARG abundances within a given environment, but also their potential mobility, hence their ability to spread to human pathogenic bacteria needs to be quantified. We developed a novel, sequencing-independent method for assessing the linkage of an ARG to a mobile genetic element by statistical analysis of multiplexed droplet digital PCR (ddPCR) carried out on environmental DNA sheared into defined, short fragments. This allows quantifying the physical linkage between specific ARGs and mobile genetic elements, here demonstrated for the sulfonamide ARG sul1 and the Class 1 integron integrase gene intI1. The method's efficiency is demonstrated using mixtures of model DNA fragments with either linked and unlinked target genes: Linkage of the two target genes can be accurately quantified based on high correlation coefficients between observed and expected values (R2) as well as low mean absolute errors (MAE) for both target genes, sul1 (R2 = 0.9997, MAE = 0.71%, n = 24) and intI1 (R2 = 0.9991, MAE = 1.14%, n = 24). Furthermore, we demonstrate that adjusting the fragmentation length of DNA during shearing allows controlling rates of false positives and false negative detection of linkage. The presented method allows rapidly obtaining reliable results in a labor- and cost-efficient manner.202336941120
367450.9966New Estimation of Antibiotic Resistance Genes in Sediment Along the Haihe River and Bohai Bay in China: A Comparison Between Single and Successive DNA Extraction Methods. Sediment is thought to be a vital reservoir for antibiotic resistance genes (ARGs). Often, studies describing and comparing ARGs and their potential hosts in sediment are based on single DNA extractions. To date, however, no study has been conducted to assess the influence of DNA extraction efficiency on ARGs in sediment. To determine whether the abundance of ARGs is underestimated, we performed five successive extraction cycles with a widely used commercial kit in 10 sediment samples collected from the Haihe River and Bohai Bay. Our results showed that accumulated DNA yields after five extractions were 1.8-3.1 times higher than that by single DNA extractions. High-throughput sequencing showed that insufficient DNA extraction could generate PCR bias and skew community structure characterization in sediment. The relative abundances of some pathogenic bacteria, such as Enterobacteriales, Lactobacillales, and Streptomycetales, were significantly different between single and successive DNA extraction samples. In addition, real-time fluorescent quantitative PCR (qPCR) showed that ARGs, intI1, and 16S rRNA gene abundance strongly increased with increasing extraction cycles. Among the measured ARGs, sulfonamide resistance genes and multidrug resistance genes were dominant subtypes in the study region. Nevertheless, different subtypes of ARGs did not respond equally to the additional extraction cycles; some continued to have linear growth trends, and some tended to level off. Additionally, more correlations between ARGs and bacterial communities were observed in the successive DNA extraction samples than in the single DNA extraction samples. It is suggested that 3-4 additional extraction cycles are required in future studies when extracting DNA from sediment samples. Taken together, our results highlight that performing successive DNA extractions on sediment samples optimizes the extractable DNA yield and can lead to a better picture of the abundance of ARGs and their potential hosts in sediments.202134616375
771360.9966Metagenome analyses of corroded concrete wastewater pipe biofilms reveal a complex microbial system. BACKGROUND: Concrete corrosion of wastewater collection systems is a significant cause of deterioration and premature collapse. Failure to adequately address the deteriorating infrastructure networks threatens our environment, public health, and safety. Analysis of whole-metagenome pyrosequencing data and 16S rRNA gene clone libraries was used to determine microbial composition and functional genes associated with biomass harvested from crown (top) and invert (bottom) sections of a corroded wastewater pipe. RESULTS: Taxonomic and functional analysis demonstrated that approximately 90% of the total diversity was associated with the phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The top (TP) and bottom pipe (BP) communities were different in composition, with some of the differences attributed to the abundance of sulfide-oxidizing and sulfate-reducing bacteria. Additionally, human fecal bacteria were more abundant in the BP communities. Among the functional categories, proteins involved in sulfur and nitrogen metabolism showed the most significant differences between biofilms. There was also an enrichment of genes associated with heavy metal resistance, virulence (protein secretion systems) and stress response in the TP biofilm, while a higher number of genes related to motility and chemotaxis were identified in the BP biofilm. Both biofilms contain a high number of genes associated with resistance to antibiotics and toxic compounds subsystems. CONCLUSIONS: The function potential of wastewater biofilms was highly diverse with level of COG diversity similar to that described for soil. On the basis of the metagenomic data, some factors that may contribute to niche differentiation were pH, aerobic conditions and availability of substrate, such as nitrogen and sulfur. The results from this study will help us better understand the genetic network and functional capability of microbial members of wastewater concrete biofilms.201222727216
773770.9966Distinctive signatures of pathogenic and antibiotic resistant potentials in the hadal microbiome. BACKGROUND: Hadal zone of the deep-sea trenches accommodates microbial life under extreme energy limitations and environmental conditions, such as low temperature, high pressure, and low organic matter down to 11,000 m below sea level. However, microbial pathogenicity, resistance, and adaptation therein remain unknown. Here we used culture-independent metagenomic approaches to explore the virulence and antibiotic resistance in the hadal microbiota of the Mariana Trench. RESULTS: The results indicate that the 10,898 m Challenger Deep bottom sediment harbored prosperous microbiota with contrasting signatures of virulence factors and antibiotic resistance, compared with the neighboring but shallower 6038 m steep wall site and the more nearshore 5856 m Pacific basin site. Virulence genes including several famous large translocating virulence genes (e.g., botulinum neurotoxins, tetanus neurotoxin, and Clostridium difficile toxins) were uniquely detected in the trench bottom. However, the shallower and more nearshore site sediment had a higher abundance and richer diversity of known antibiotic resistance genes (ARGs), especially for those clinically relevant ones (e.g., fosX, sul1, and TEM-family extended-spectrum beta-lactamases), revealing resistance selection under anthropogenic stresses. Further analysis of mobilome (i.e., the collection of mobile genetic elements, MGEs) suggests horizontal gene transfer mediated by phage and integrase as the major mechanism for the evolution of Mariana Trench sediment bacteria. Notably, contig-level co-occurring and taxonomic analysis shows emerging evidence for substantial co-selection of virulence genes and ARGs in taxonomically diverse bacteria in the hadal sediment, especially for the Challenger Deep bottom where mobilized ARGs and virulence genes are favorably enriched in largely unexplored bacteria. CONCLUSIONS: This study reports the landscape of virulence factors, antibiotic resistome, and mobilome in the sediment and seawater microbiota residing hadal environment of the deepest ocean bottom on earth. Our work unravels the contrasting and unique features of virulence genes, ARGs, and MGEs in the Mariana Trench bottom, providing new insights into the eco-environmental and biological processes underlying microbial pathogenicity, resistance, and adaptative evolution in the hadal environment.202235468809
683480.9965Landscape of antibiotic resistance genes and bacterial communities in groundwater on the Tibetan Plateau, and distinguishing their difference with low-altitude counterparts. Groundwater is a vital source of drinking water for Tibetans. Antibiotic resistance genes (ARGs) and bacterial communities in groundwater on the Tibetan Plateau remain unclear. Furthermore, the characterization of their differences between high-altitude and low-altitude groundwater is still unrevealed. Herein, 32 groundwater samples were collected on the plateau, and intra- and extracellular ARGs (iARGs and eARGs), and bacterial communities were characterised through qPCR assays to 19 ARGs and 16S rRNA sequencing. It showed top four abundant intra- and extracellular last-resort ARGs (LARGs) were bla(OXA-48), mcr-1, vanA, and vanB, whereas dominant common ARGs (CARGs) were tetA and ermB, respectively. CARGs had higher abundances than LARGs, and iARGs were more frequently detected than eARGs. Proteobacteria, an invasive resident phylum, and Firmicutes dominated eDNA release. Network analysis revealed all observed LARGs co-occurred with pathogenic and non-pathogenic bacteria. Community diversity was significantly associated with longitude and elevation, while nitrate correlated with ARGs. Comparative analysis demonstrated eARG frequencies and abundances were higher at high altitudes than at low altitudes. Additionally, Acinetobacter and Pseudomonas specifically dominated at high altitudes. This study reveals the widespread prevalence of ARGs, particularly LARGs, in groundwater on the less-disturbed Tibetan Plateau and underlines the potential risks associated with the LARG-carrying bacteria. ENVIRONMENTAL IMPLICATION: Antibiotic resistance genes (ARGs), which are defined as emerging environmental contaminants, are becoming a global concern due to their ability to confer antibiotic resistance to pathogens. Our findings highlight the prevalence of ARGs, particularly LARGs, in groundwater on the Tibetan Plateau, and the possibility that naturally-occurring pathogenic and non-pathogenic bacteria carry multiple LARGs. In addition, we further reveal differences in the distribution of ARGs and bacterial community between high-altitude and low-altitude groundwater. Collectively, our findings offer an important insight into the potential public risks related to groundwater on the Tibetan Plateau.202337595466
683690.9965Microbiome and antibiotic resistome in household dust from Beijing, China. We spend ever-increasing time indoors along with urbanization; however, the geographical distribution patterns of microbiome and antibiotic resistome, and their driving forces in household environment remains poorly characterized. Here, we surveyed the bacterial and fungal communities, and the resistome in settled dust gathered from 82 homes located across Beijing, China, employing Illumina sequencing and high-throughput quantitative PCR techniques. There was no clear geographical distribution pattern in dust-related bacterial communities although a slight but significant (P < 0.05) distance-decay relationship occurred in its community similarity; by contrast, a relatively distinct geographical clustering and a stronger distance-decay relationship were observed in fungal communities at the local scale. The cross-domain (bacteria versus fungi) relationships in the microbiome of the dust samples were mostly observed as robust co-occurrence correlations. The bacterial communities were dominated by Proteobacteria and Actinobacteria phyla, with human skin, soil and plants being potential major sources. The fungal communities largely comprised potential allergens (a median 61% of the fungal sequences), with Alternaria genus within Ascomycota phylum being the most predominant taxa. The profile of dust-related bacterial communities was mainly affected by housing factors related to occupants and houseplants, while that of fungal communities was determined by georeferenced environmental factors, particularly vascular plant diversity. Additionally, a great diversity (1.96 on average for Shannon index) and normalized abundance (2.22 copies per bacterial cell on average) of antibiotic resistance genes were detected across the dust samples, with the dominance of genes resistant to vancomycin and Macrolide-Lincosamide-Streptogramin B. The resistome profile exhibited no distinct geographical pattern, and was primarily driven by certain bacterial phyla and occupancy-related factors. Overall, we underline the significance of anthropogenic impacts and house location in structuring bacterial and fungal communities inside homes, respectively, and suggest that household dust is an overlooked reservoir for antibiotic resistance.202032248025
7723100.9965Karst spring microbiome: Diversity, core taxa, and community response to pathogens and antibiotic resistance gene contamination. Karst aquifers are important water resources for drinking water supplies worldwide. Although they are susceptible to anthropogenic contamination due to their high permeability, there is a lack of detailed knowledge on the stable core microbiome and how contamination may affect these communities. In this study, eight karst springs (distributed across three different regions in Romania) were sampled seasonally for one year. The core microbiota was analysed by 16S rRNA gene amplicon sequencing. To identify bacteria carrying antibiotic resistance genes and mobile genetic elements, an innovative method was applied, consisting of high-throughput antibiotic resistance gene quantification performed on potential pathogen colonies cultivated on Compact Dry™ plates. A taxonomically stable bacterial community consisting of Pseudomonadota, Bacteroidota, and Actinomycetota was revealed. Core analysis reaffirmed these results and revealed primarily freshwater-dwelling, psychrophilic/psychrotolerant species affiliated to Rhodoferax, Flavobacterium, and Pseudomonas genera. Both sequencing and cultivation methods indicated that more than half of the springs were contaminated with faecal bacteria and pathogens. These samples contained high levels of sulfonamide, macrolide, lincosamide and streptogramins B, and trimethoprim resistance genes spread primarily by transposase and insertion sequences. Differential abundance analysis found Synergistota, Mycoplasmatota, and Chlamydiota as suitable candidates for pollution monitoring in karst springs. This is the first study highlighting the applicability of a combined approach based on high-throughput SmartChip™ antibiotic resistance gene quantification and Compact Dry™ pathogen cultivation for estimating microbial contaminants in karst springs and other challenging low biomass environments.202337364839
3538110.9965Amoxicillin Increased Functional Pathway Genes and Beta-Lactam Resistance Genes by Pathogens Bloomed in Intestinal Microbiota Using a Simulator of the Human Intestinal Microbial Ecosystem. Antibiotics are frequently used to treat bacterial infections; however, they affect not only the target pathogen but also commensal gut bacteria. They may cause the dysbiosis of human intestinal microbiota and consequent metabolic alterations, as well as the spreading of antibiotic resistant bacteria and antibiotic resistance genes (ARGs). In vitro experiments by simulator of the human intestinal microbial ecosystem (SHIME) can clarify the direct effects of antibiotics on different regions of the human intestinal microbiota, allowing complex human microbiota to be stably maintained in the absence of host cells. However, there are very few articles added the antibiotics into this in vitro model to observe the effects of antibiotics on the human intestinal microbiota. To date, no studies have focused on the correlations between the bloomed pathogens caused by amoxicillin (AMX) exposure and increased functional pathway genes as well as ARGs. This study investigated the influence of 600 mg day(-1) AMX on human intestinal microbiota using SHIME. The impact of AMX on the composition and function of the human intestinal microbiota was revealed by 16S rRNA gene sequencing and high-throughput quantitative PCR. The results suggested that: (i) AMX treatment has tremendous influence on the overall taxonomic composition of the gut microbiota by increasing the relative abundance of Klebsiella [linear discriminant analysis (LDA) score = 5.26] and Bacteroides uniformis (LDA score = 4.75), as well as taxonomic diversity (Simpson, P = 0.067, T-test; Shannon, P = 0.061, T-test), and decreasing the members of Parabacteroides (LDA score = 4.18), Bifidobacterium (LDA score = 4.06), and Phascolarctobacterium (LDA score = 3.95); (ii) AMX exposure significantly enhanced the functional pathway genes and beta-lactam resistance genes, and the bloomed pathogens were strongly correlated with the metabolic and immune system diseases gene numbers (R = 0.98, P < 0.001) or bl2_len and bl2be_shv2 abundance (R = 0.94, P < 0.001); (iii) the changes caused by AMX were "SHIME-compartment" different with more significant alteration in ascending colon, and the effects were permanent, which could not be restored after 2-week AMX discontinuance. Overall results demonstrated negative side-effects of AMX, which should be considered for AMX prescription.202032582117
6972120.9965The antibiotic resistome of free-living and particle-attached bacteria under a reservoir cyanobacterial bloom. In freshwater systems, both antibiotic resistance genes (ARGs) and cyanobacterial blooms attract global public health concern. Cyanobacterial blooms can greatly impact bacterial taxonomic communities, but very little is known about the influence of the blooms on antibiotic resistance functional community. In this study, the ARGs in both free-living (FL) and particle-attached (PA) bacteria under bloom and non-bloom conditions were simultaneously investigated in a subtropical reservoir using high-throughput approaches. In total, 145 ARGs and 9 mobile genetic elements (MGEs) were detected. The most diverse and dominant of which (68.93%) were multidrug resistance genes and efflux pump mechanism. The richness of ARGs in both FL and PA bacteria was significantly lower during the bloom period compared with non-bloom period. The abundance of ARGs in FL bacteria was significantly lower under bloom condition than in the non-bloom period, but the abundance of ARGs in PA bacteria stayed constant. More importantly, the resistant functional community in PA bacteria was more strongly influenced by the cyanobacterial bloom than in the FL bacteria, although >96% ARGs were shared in both FL and PA bacteria or both bloom and non-bloom periods. We also compared the community compositions between taxonomy and function, and found antibiotic resistant communities were highly variable and exhibited lower similarity between bloom and non-bloom periods than seen in the taxonomic composition, with an exception of FL bacteria. Altogether, cyanobacterial blooms appear to have stronger inhibitory effect on ARG abundance in FL bacteria, and stronger influence on antibiotic resistant community composition in PA bacteria. Our results further suggested that both neutral and selective processes interactively affected the ARG composition dynamics of the FL and PA bacteria. However, the antibiotic resistant community of FL bacteria exhibited a higher level of temporal stochasticity following the bloom event than PA bacteria. Therefore, we emphasized the bacterial lifestyles as an important mechanism, giving rise to different responses of antibiotic resistant community to the cyanobacterial bloom.201829734061
3116130.9965Prediction of Antibiotic Resistance Genes in Cyanobacterial Strains by Whole Genome Sequencing. Cyanobacteria are ubiquitous in freshwater environments, but their role in aquatic resistome remains unclear. In this work, we performed whole genome sequencing on 43 cyanobacterial strains isolated from Portuguese fresh/wastewaters. From 43 available non-axenic unicyanoabacterial cultures (containing only one cyanobacterial strain and their co-occurring bacteria), it was possible to recover 41 cyanobacterial genomes from the genomic assemblies using a genome binning software, 26 of which were classified as high-quality based on completeness, contamination, N50 and contig number thresholds. By using the comprehensive antibiotic resistance database (CARD) on the assembled samples, we detected four antibiotic resistance gene (ARG) variants, conferring resistance in pathogenic bacteria to tetracyclines, fluoroquinolones (adeF-type) and macrolides (ermF-type, mefC-type and mphG-type). Among these, adeF-type was the most prevalent gene, found across 11 cyanobacterial genomes from the Nostocales order. Planktothrix presented the highest variety of close ARG matches, with hits for the macrolide resistance genes ermF-type, mefC-type and mphG-type. An analysis of the genomic assemblies also revealed an additional 12 ARGs in bacteria from the phyla Firmicutes, Proteobacteria and Bacteroidetes, present in the cyanobacterial cultures, foreseeing the horizontal gene transfer of ARGs with cyanobacteria. Additionally, more than 200 partial ARGs were detected on each recovered cyanobacterial genome, allowing for future studies of antibiotic resistance genotype/phenotype in cyanobacteria. These findings highlight the importance of further efforts to understand the role of cyanobacteria on the aquatic resistome from a One Health perspective.202540572139
7691140.9964Antimicrobial Chemicals Associate with Microbial Function and Antibiotic Resistance Indoors. Humans purposefully and inadvertently introduce antimicrobial chemicals into buildings, resulting in widespread compounds, including triclosan, triclocarban, and parabens, in indoor dust. Meanwhile, drug-resistant infections continue to increase, raising concerns that buildings function as reservoirs of, or even select for, resistant microorganisms. Support for these hypotheses is limited largely since data describing relationships between antimicrobials and indoor microbial communities are scant. We combined liquid chromatography-isotope dilution tandem mass spectrometry with metagenomic shotgun sequencing of dust collected from athletic facilities to characterize relationships between indoor antimicrobial chemicals and microbial communities. Elevated levels of triclosan and triclocarban, but not parabens, were associated with distinct indoor microbiomes. Dust of high triclosan content contained increased Gram-positive species with diverse drug resistance capabilities, whose pangenomes were enriched for genes encoding osmotic stress responses, efflux pump regulation, lipid metabolism, and material transport across cell membranes; such triclosan-associated functional shifts have been documented in laboratory cultures but not yet from buildings. Antibiotic-resistant bacterial isolates were cultured from all but one facility, and resistance often increased in buildings with very high triclosan levels, suggesting links between human encounters with viable drug-resistant bacteria and local biocide conditions. This characterization uncovers complex relationships between antimicrobials and indoor microbiomes: some chemicals elicit effects, whereas others may not, and no single functional or resistance factor explained chemical-microbe associations. These results suggest that anthropogenic chemicals impact microbial systems in or around buildings and their occupants, highlighting an emergent need to identify the most important indoor, outdoor, and host-associated sources of antimicrobial chemical-resistome interactions. IMPORTANCE The ubiquitous use of antimicrobial chemicals may have undesired consequences, particularly on microbes in buildings. This study shows that the taxonomy and function of microbes in indoor dust are strongly associated with antimicrobial chemicals-more so than any other feature of the buildings. Moreover, we identified links between antimicrobial chemical concentrations in dust and culturable bacteria that are cross-resistant to three clinically relevant antibiotics. These findings suggest that humans may be influencing the microbial species and genes that are found indoors through the addition and removal of particular antimicrobial chemicals.201830574558
7814150.9964Combination of flow cytometry and molecular analysis to monitor the effect of UVC/H(2)O(2) vs UVC/H(2)O(2)/Cu-IDS processes on pathogens and antibiotic resistant genes in secondary wastewater effluents. The efficiency of a new Advanced Oxidation Process (AOP), namely the photo Fenton like process UV-C/H(2)O(2)/IDS-Cu, in removing determinants of antibiotic resistance and pathogenic bacteria was compared to a consolidated AOP (namely UV-C/H(2)O(2)) in a secondary treated municipal WasteWater (WW). A reductionist experimental laboratory-based approach was applied on real WW and the parameters were collected by an alternative integrated approach using (i) flow cytometry to enumerate bacteria and test for the fitness of the bacterial communities and (ii) molecular analyses to define the community composition (16S rRNA amplicon sequencing) and the abundances of Antibiotic Resistance Genes (ARGs) and of the class 1 integron (intI1 gene) (by quantitative PCR). The same approach was applied also to post-treatment regrowth tests (24 h) to define the potential persistence of the tested parameters. These experiments were performed in both, human pathogens favorable conditions (HPC, in rich medium and 37°C) and in environmental mimicking conditions (EMC, original WW and 20°C). UV-C/H(2)O(2)/IDS-Cu process resulted to be more effective than the UV-C/H(2)O(2)in inactivating bacterial cells in the EMC post-treatment regrowth experiments. Both AOPs were efficiently abating potential human pathogenic bacteria and ARGs in the HPC regrowth experiments, although this trend could not be detected in the measurements taken immediately after the disinfection. In comparison with the UV-C/H(2)O(2), the UV-C/H(2)O(2)/IDS-Cu process did not apparently offer significant improvements in the abatement of the tested parameters in the WW effluent but, by evaluating the results of the regrowth experiments it was possible to extrapolate more complex trends, suggesting contrasting efficiencies visible only after a few hours. This study offers a detailed view on the abatement efficiency of microbiological/genetic parameters for the UV-C/H(2)O(2)/IDS-Cu process, calling for technical adjustments for this very promising technology. At the same time, our results clearly demonstrated the inadequacy of currently applied methodologies in the evaluation of specific parameters (e.g. determinants of antibiotic resistance and pathogenic bacteria) in WW.202032711221
3321160.9964Saccharomyces boulardii CNCM I-745 supplementation modifies the fecal resistome during Helicobacter pylori eradication therapy. BACKGROUND: The gut microbiota is a significant reservoir of antimicrobial resistance genes (ARGs). The use and misuse of antimicrobials can select multi-resistant bacteria and modify the repertoire of ARGs in the gut. Developing effective interventions to manipulate the intestinal resistome would allow us to modify the antimicrobial resistance risk. MATERIALS AND METHODS: Applying shotgun metagenomics, we compared the composition of fecal resistome from individuals treated with triple therapy for Helicobacter pylori plus Saccharomyces boulardii CNCM-I 745 (Sb) versus triple antibiotherapy without S. boulardii (control) before, after, and one month after treatments. DNA samples were sequenced on an Illumina NovaSeq 6000 platform. Reads were trimmed and filtered for quality, and the reads classified as host genome were removed from further analysis. We used the ResFinder database for resistome analysis and the web-based tool ResistoXplorer and RStudio for graphical representation and statistical analysis. RESULTS: We identified 641 unique ARGs in all fecal samples, conferring resistance to 18 classes of antibiotics. The most prevalent ARGs found in at least 90% of the samples before the treatments were against tetracyclines, MLS-B (macrolide, lincosamide, and streptogramin B), beta-lactams, and aminoglycosides. Differential abundance analysis allowed the identification of ARGs significantly different between treatment groups. Thus, immediately after the treatments, the abundance of ARGs that confer resistance to lincosamides, tetracyclines, MLS-B, and two genes in the beta-lactam class (cfxA2 and cfxA3) was significantly lower in the group that received Sb than in the control group (edgeR, FDR <0.05). CONCLUSION: Our study demonstrated that the addition of S. boulardii CNCM-I 745 to the conventional antibiotic eradication therapy for H. pylori reduced the abundance of ARGs, particularly those genes that confer resistance to lincosamides, tetracyclines, MLS-B, and a few genes in the beta-lactams class.202234990038
6899170.9964Cyanobacteria mediate the dissemination of bacterial antibiotic resistance through conjugal transfer. Cyanobacterial blooms are expanding world-wide in freshwater and marine environments, and can cause serious ecological and environmental issues, which also contribute to the spread of antibiotic resistance genes (ARGs). However, the mechanistic understanding of cyanobacteria-mediated resistance dynamics is not fully elucidated yet. We selected Microcystis aeruginosa as a model cyanobacteria to illustrate how cyanobacteria mediate the evolution and transfer processes of bacterial antibiotic resistance. The results show that the presence of cyanobacteria significantly decreased the abundance of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) by 3%-99% and 2%-18%, respectively. In addition, it clearly altered bacterial community structure, with the dominant genera evolving from Acinetobacter (27%) and Enterobacter (42%) to Porphyrobacter (59%). The abundance of ARGs positively correlated with Proteobacteria and Firmicutes, rather than Cyanobacteria, and Bacteroidetes. In the presence of cyanobacteria, the transfer events of bacterial resistance genes via conjugation were found to decrease by 10%-89% (p < 0.05). Surprisingly, we found an extradentary high transfer frequency (about 0.1) for the ARGs via plasmid conjugation from the bacteria into M. aeruginosa population. It confirmed the role of cyanobacterial population as the competent hosts to facilitate ARGs spreading. Our findings provide valuable information on the risk evaluation of ARGs caused by cyanobacterial blooms in aquatic environments, key for the protection and assessment of aquatic environmental quality.202439047887
7374180.9964Unravelling the Portuguese Coastal and Transitional Waters' Microbial Resistome as a Biomarker of Differential Anthropogenic Impact. Portugal mainland and Atlantic archipelagos (Madeira and Azores) provide a wide array of coastal ecosystems with varying typology and degrees of human pressure, which shape the microbial communities thriving in these habitats, leading to the development of microbial resistance traits. The samples collected on the Portuguese northeast Atlantic coast waters show an unequivocal prevalence of Bacteria over Archaea with a high prevalence of Proteobacteria, Cyanobacteria, Bacteroidetes and Actinobacteria. Several taxa, such as the Vibrio genus, showed significant correlations with anthropogenic pollution. These anthropogenic pressures, along with the differences in species diversity among the surveyed sites, lead to observed differences in the presence and resistance-related sequences' abundance (set of all metal and antibiotic resistant genes and their precursors in pathogenic and non-pathogenic bacteria). Gene ontology terms such as antibiotic resistance, redox regulation and oxidative stress response were prevalent. A higher number of significant correlations were found between the abundance of resistance-related sequences and pollution, inorganic pressures and density of nearby population centres when compared to the number of significant correlations between taxa abundance at different phylogenetic levels and the same environmental traits. This points towards predominance of the environmental conditions over the sequence abundance rather than the taxa abundance. Our data suggest that the whole resistome profile can provide more relevant or integrative answers in terms of anthropogenic disturbance of the environment, either as a whole or grouped in gene ontology groups, appearing as a promising tool for impact assessment studies which, due to the ubiquity of the sequences across microbes, can be surveyed independently of the taxa present in the samples.202236287893
7133190.9964Prevalence of antibiotic resistance genes in bacterial communities associated with Cladophora glomerata mats along the nearshore of Lake Ontario. The alga Cladophora glomerata can erupt in nuisance blooms throughout the lower Great Lakes. Since bacterial abundance increases with the emergence and decay of Cladophora, we investigated the prevalence of antibiotic resistance (ABR) in Cladophora-associated bacterial communities up-gradient and down-gradient from a large sewage treatment plant (STP) on Lake Ontario. Although STPs are well-known sources of ABR, we also expected detectable ABR from up-gradient wetland communities, since they receive surface run-off from urban and agricultural sources. Statistically significant differences in aquatic bacterial abundance and ABR were found between down-gradient beach samples and up-gradient coastal wetland samples (ANOVA, Holm-Sidak test, p < 0.05). Decaying and free-floating Cladophora sampled near the STP had the highest bacterial densities overall, including on ampicillin- and vancomycin-treated plates. However, quantitative polymerase chain reaction analysis of the ABR genes ampC, tetA, tetB, and vanA from environmental communities showed a different pattern. Some of the highest ABR gene levels occurred at the 2 coastal wetland sites (vanA). Overall, bacterial ABR profiles from environmental samples were distinguishable between living and decaying Cladophora, inferring that Cladophora may control bacterial ABR depending on its life-cycle stage. Our results also show how spatially and temporally dynamic ABR is in nearshore aquatic bacteria, which warrants further research.201728192677